Quantitative assessment of choriocapillaris flow deficits and type 1 macular neovascularization growth in age-related macular degeneration

During the past 15 years, new treatment paradigms for neovascular age-related macular degeneration (nvAMD) have evolved due to the advent of intravitreal anti-vascular endothelial growth factor (VEGF) therapy and rapid advances in retinal imaging. Recent publications describe eyes with type 1 macular neovascularization (MNV) as showing more resistance to macular atrophy than eyes with other lesion types. We sought to explore whether the perfusion status of the native choriocapillaris (CC) surrounding type 1 MNV influences its pattern of growth. To evaluate this effect, we analyzed a case series of 22 eyes from 19 nvAMD patients with type 1 MNV exhibiting growth on swept-source optical coherence tomography angiography (SS-OCTA) over a minimum follow-up of 12 months. We observed an overall weak correlation between type 1 MNV growth and CC flow deficits (FDs) average size (τ = 0.17, 95% CI [− 0.20, 0.62]) and a moderate correlation with CC FD % (τ = 0.21, 95% CI [− 0.16, 0.68]). Type 1 MNV was located beneath the fovea in most of the eyes (86%) and median visual acuity was 20/35 Snellen equivalent. Our results support that type 1 MNV recapitulates areas of CC blood flow impairment while serving to preserve foveal function.


Discussion
In our analysis of 22 type 1 MNV identified in 19 patients with nvAMD, we observed a monotonic trend between neovascularization growth and CC FD average size and %. If a particular region showed increased CC FD size or %, it was likely to observe neovascular growth in that direction over follow-up. This trend was observed even in cases in which the correlation between neovascular growth and FD features did not reach statistical significance. These results suggest that there may be a trend between type 1 MNV growth directionality and nearby CC flow impairment. This pattern was observed irrespective of the location of the neovascularization (sub-foveal vs parafoveal). If the location relative to the foveal center was determinant, one would expect growth directionality to be different between sub-foveal and parafoveal type 1 MNV, but this was not observed. While we cannot discount the possibility that our findings are associated with other explanatory variables, we believe our results suggest that CC flow alterations influence the pattern of type 1 MNV growth in nvAMD.
Our findings are consistent with those of prior investigations suggesting that type 1 MNV may recapitulate areas of CC flow impairment 22 . We observed that, after a mean follow-up of 28 ± 3 months, 86% of the eyes showed type 1 MNV under the central fovea and relative preservation of the visual function (median BCVA: 0.25; interquartile range, 0.2-0.4 logMAR). All the patients received treatment during the follow-up, albeit with different treatment intervals. The herein reported functionality agrees with the data reported from several large datasets and supports that type 1 MNV might be able to support RPE and photoreceptor cells 5,9,23 .
Our findings might have some implications regarding the pathophysiologic processes of nvAMD. Some authors have noted that CC FDs' average size in the central macula increases with aging, and is augmented in the fellow eyes of patients with nvAMD and in eyes that progress to MNV and cRORA [12][13][14][15]17 . In eyes with nvAMD, Moult et al. have shown non-uniformity of CC flow impairment surrounding type 1 MNV and suggested that angle-dependent and lesion-centered analyses would be appropriate for longitudinal studies 16 . Recently, Corvi et al. observed that in eyes with intermediate AMD that progressed to type 1 or 2 MNV there was focal impairment of the CC, contrary to eyes progressing to type 3 MNV or cRORA, in which the impairment was diffuse 14 . While our study corroborates those observations, we add additional information that may be useful for a better understanding of the association between neovascular growth and CC blood flow impairment.
We observed that CC FD features (size and %) were not uniform in the 600 µm ring area surrounding type 1 MNV, and we verified a trend of MNV growth over areas with increased average size and % of FD. These findings suggest there might be a perfusion-dependent expression of angiogenic factors driving neovascularization growth towards areas with larger FDs. While this neovascular growth pattern reached statistical significance in 70% of the cases, in the remainder, an association could only be perceived in half of the region surrounding the www.nature.com/scientificreports/ MNV and lowered the averaged correlation coefficients representing the entire cohort. We acknowledge that other factors potentially associated with growth directionality should be explored and might have accounted for these findings, including resistance in choroidal venous outflow and the type of deposits at the level of the RPE complex. We believe this topic should be further studied as it is likely to expand our understanding of the mechanisms involved in some MNV processes.
The main strengths of this study are long clinical follow-up and precision in the image analysis. To the best of our knowledge, this is the first study to assess the association between MNV growth patterns and CC flow analysis with a longitudinal design. The mean follow-up interval between the baseline and final SS-OCTA was 28 ± 3 months, which was greater than our predicted minimal timeframe (1 year) needed to observe significant changes in neovascular surface area growth on SS-OCTA 20 . Image analysis was based on a rigorous protocol that included semi-automatic segmentation of the retinal layers of interest in each B-scan and automated algorithms previously validated to perform registration of subsequent SS-OCTA acquisitions and to segment FDs and neovascular blood flow 10,24,25 . This study was limited by the small sample size and limitations inherent to its retrospective design, which did not enable a uniform follow-up in all cases nor control diurnal variation or arterial pressure effects on the choriocapillaris. Although most patients had yearly SS-OCTA meeting inclusion criteria, in 9 cases (38%) the interval between SS-OCTA ranged between 2 and 3 years. Analyzing yearly SS-OCTA in these cases could have impacted the strength of the association between neovascular growth and FD quantitative measurements, albeit it is unlikely that it would have changed the monotonic association hereby reported. We also did not distinguish between different AMD subgroups (exudative vs non-exudative; treatment naive vs receiving treatment) given the low number of eyes per putative subgroup. Selecting cases with neovascular growth over follow-up also impacts www.nature.com/scientificreports/ the generalization of our results. We believe that evaluating FD features after type 1 MNV reaches a stable size can inform on the impact of choriocapillaris changes in neovascularization growth in future studies. We also acknowledge that the mean age of our cohort was 76 ± 2 years and 84% of the patients had systemic hypertension well controlled with medical treatment. As aging and systemic hypertension have been associated with an increased size of FD, we cannot exclude a contributory role. Additionally, the current study used non-averaged acquisitions performed by an SS-OCTA system. Although SS-OCTA is the more reliable technology for in vivo visualization of the CC, limitations inherent to its lateral resolution, sampling rate, and speckle noises must be acknowledged 10 . One strategy to mitigate the influence of speckle noises in CC quantification is using multiplescan averaging. However, due to the study design, we used single scans for CC analysis, which is associated with an estimated mean bias of 4.5% 10 .
In conclusion, we observed a positive trend between the growth patterns of type 1 MNV and two surrogate markers of CC blood flow impairment, i.e., increased FD average size and FD %. Recapitulation of CC areas with blood flow impairment was associated with preserved visual function, which supports that type 1 MNV might be a compensatory process for restoring nutritional support to the macula, eventually becoming a highly effective AMD treatment capable of preserving central vision.

Methods
A retrospective analysis of a consecutive case series of patients with neovascular AMD seen at regular intervals for routine monitoring and/or treatment with intravitreal anti-VEGF therapy by one retinal specialist (K.B.F) was performed at Vitreous Retina Macula Consultants of New York (New York, USA) between July and December 2021. This study was approved by the Western Institutional Review Board (Olympia, WA), written informed www.nature.com/scientificreports/ consent was obtained from each participant, and all methods were performed in accordance with the declaration of Helsinki. Inclusion criteria consisted of patients with nvAMD managed on a treat and extend (TER) 26 dosing regimen of anti-VEGF therapy in whom growth of type 1 MNV was documented with SS-OCTA with at least one 1-year interval. 1-year intervals were predicted to be sufficient to observe the evolution of treated type 1 MNV 20 . Baseline was defined as the first visit during which SS-OCTA identified type 1 MNV flow signal with a greatest linear dimension of ≥ 250 µm or 0.2 mm due to the difficulty of measuring these lesions reproducibly 21 . Since TER injection intervals ranged from 4 to 10 weeks, the visits closest to 12 months since the baseline or prior 1-year time point were included in the analyses. When 2 eyes of the same patient met eligibility, data from both eyes were included. Type 1 MNV was defined according to the Consensus Nomenclature for Reporting Neovascular Age-Related Macular Degeneration Data (CONAN) criteria 3 . Exclusion criteria included: previous treatment in the study eye with photodynamic therapy, high myopia (≥ − 6.0 diopters), a history of any other retinal disease deemed to affect SS-OCTA, complete retinal pigment epithelium and outer retinal atrophy (cRORA) 27 ; a fibrovascular pigment epithelium detachment height > 250 µm; media opacities interfering with retinal imaging, eyes with any evidence of type 2 or 3 MNV, eyes with multiple type 1 MNV lesions, and eyes with neovascular lesions within 600 µm of the border of the SS-OCTA scan area.
All patients had undergone complete ophthalmologic examinations, including measurement of the bestcorrected visual acuity (BCVA) using Snellen charts, slit-lamp biomicroscopy, indirect fundus ophthalmoscopy, structural SD-OCT (Spectralis HRA + OCT2 (Heidelberg Engineering, Heidelberg, Germany)) and SS-OCTA (PLEX Elite 9000 SS-OCT (Carl Zeiss Meditec, Inc, Dublin, CA)). The technical specifications of the SS-OCT For each eye, the SS-OCTA data analyzed was 6 × 6 mm (500 A-scans × 500 B-scans) SS-OCTA acquisitions centered on the fovea with a minimum signal strength index of 8, and minimal motion artifacts. SS-OCTA acquisitions from cases meeting inclusion criteria were exported as raw data (.IMG files) for image analysis. Prior to grading, SS-OCTA files were subjected to a preliminary review to identify artifacts (for example motion artifacts, signal shadowing due to intravitreal suspensions, etc.) affecting choriocapillaris imaging that would preclude reliable analysis. In cases considered potential candidates for an analysis of the choriocapillaris flow deficits, the segmentation of Bruch's membrane was automatically performed by combined use of SS-OCT and SS-OCTA cubes and manually corrected if necessary, and the files were pre-processed to obtain an improved visualization and assessment of the choriocapillaris, as previously described 10 . Subsequently, SS-OCTA of subsequent visits was aligned with the baseline acquisition using an automated non-rigid registration algorithm, as previously described 10,24 .
SS-OCTA was processed to extract the neovascular contour outline and to segment choriocapillaris flow deficits. Image processing, image analysis, and statistical analysis were done using a code designed in MAT-LAB version R2020a (The MathWorks Inc., Natick, MA, USA). The neovascular contour was obtained using a previously described and validated automated algorithm that detected angiographic flow within the outer retina to the choriocapillaris (ORCC) slab after the removal of vessel projection artifacts 25 . An en face image of the choriocapillaris was obtained using a 16-µm thick slab with its anterior boundary located 4 µm beneath the Bruch's membrane. Subsequently, flow deficits were segmented using a previously described and validated www.nature.com/scientificreports/ methodology 28,29 . The resulting neovascularization segmentation outlines were analyzed by two retina specialists. Cases in which there was disagreement on the MNV segmentation outline were excluded. A graphical representation of the different image processing steps is shown in supplemental Fig. 1. Following previous conventions for the assessment of CC FD features in the vicinity of MNV 21 , a quantitative analysis of CC FD was conducted and correlated with neovascular growth over follow-up within a 600µm region extending from the neovascular contour in baseline scans. This analysis was performed using radial sectors centered on the MNV centroid, as previously postulated 16 . Two quantitative metrics were assessed: the CC FD percentage (%) and the average CC FD area. The CC FD% was defined as the percentage of pixels representing flow deficits relative to all the pixels within a sector. The average CC FD area within a sector was given in square millimeters (mm 2 ). Neovascular growth was defined as the average difference in neovascularization outlines within a sector between visits spaced over more than 1 year and given in mm 2 . Neovascular growth and the corresponding values of FD features (% and average size) were computed for 23 sectors with 50% overlap, i.e. sectors measuring 30º (central angle) and overlapping half of adjacent sectors. Overlapping adjacent sectors enabled to mitigate the bias associated with iatrogenic FDs features, i.e., FDs spanned by the intersectoral borders, which has been considered a limitation of radial sectors analysis 16 . A graphical representation of the image analysis protocol is depicted in supplemental Fig. 2. The distribution of the variables of interest in the entire cohort was assessed using data plots. Before data plotting, the presence of outliers was assessed and removed using the isoutlier function for MATLAB. Afterward, FD features data was binned in uniform classes following the square root choice 30 , and the average neovascular growth for each interval (mean± standard error of the mean) was plotted and visually inspected. The association between FD features and neovascular growth was assessed using Kendall's rank correlation coefficient (τ) and the resulting values were interpreted as previously described (less than 0.10: very weak; 0.10 to 0.19: weak; 0.20 to 0.29: moderate; and 0.30 or above: strong correlation) 31 . A τ value was obtained for each FD feature (% and average size) for each case. A global τ average for the entire cohort was calculated using Fisher's Z transformation.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.